The non-covalent complexes of duplexes DNA and 9 ginsenosides（1 aglycone and 8 glycosides） were investigated using electrospray ionization mass spectrometry（ESI-MS） in the gas phase. The results of relative bindin...The non-covalent complexes of duplexes DNA and 9 ginsenosides（1 aglycone and 8 glycosides） were investigated using electrospray ionization mass spectrometry（ESI-MS） in the gas phase. The results of relative binding affinities in negative ion mode revealed that several factors impact on the duplexbinding properties of ginsenosides. Glycosylations of 20（S）-protopanaxadiol ginsenosides at the position C-20 and 20（S）-protopanaxatriol classification at the position C-6 enhanced the fraction of bound DNA sharply. A rhamnose moiety shows little lower binding intensities than glucose at the same position.Ginsenosides of 20（S）-protopanaxatriol result in subtle higher binding affinities toward the duplex DNA than 20（S）-protopanaxadiol family. However, glycosylation with two sugar moieties does not show a higher binding affinity than with only one moiety. The collision-induced dissociation experimental data demonstrate the gas-phase stability and fragmentation patterns of the ginsenoside/DNA complexes are related to the glycoside number. Positive ion ESI mass spectra of the complexes were also recorded. The result of ESI-MS suggests that hydrogen bonds are the dominate interaction between ginsenosides and DNA. Similar results were obtained in solution-phase by UV spectroscopy, which exhibit a hyperchromism and blue-shift effect when DNA solution was titrated by individual ginsenoside.展开更多
基于国际生命条码联盟(CBOL,the Consortium for the Barcode of Life)提出的barcoding技术所确定的序列区域,针对猪的线粒体COⅠ基因序列设计特异性引物。为了避免食品中PCR抑制剂的影响,本实验设置GCG(胰岛素受体)基因125bp的纯化片...基于国际生命条码联盟(CBOL,the Consortium for the Barcode of Life)提出的barcoding技术所确定的序列区域,针对猪的线粒体COⅠ基因序列设计特异性引物。为了避免食品中PCR抑制剂的影响,本实验设置GCG(胰岛素受体)基因125bp的纯化片段为内参照,控制假阴性结果的出现。通过优化PCR反应条件,猪和GCG基因的特异性产物在79.2℃和75℃有特异性熔解峰。设置牛、羊、鸡、兔、鼠和空白对照,均无扩增。测序结果表明,该猪特异性引物的PCR产物含有245bp的核苷酸序列与GenBank中相应序列吻合;该方法检出限为0.001%。展开更多
BACKGROUND Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver...BACKGROUND Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed. AIM To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method. METHODS A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay. RESULTS Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P<0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with markedsevere inflammation were significantly higher than those with mild-moderate inflammation (P<0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R^2=0.673, P<0.01) or plasma DNA (R^2=0.597, P<0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild–moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively. CONCLUSION The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.展开更多
The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant o...The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.展开更多
基金supported by the National Natural Science Foundation of China (Nos. 21073178 and 21175128)
文摘The non-covalent complexes of duplexes DNA and 9 ginsenosides（1 aglycone and 8 glycosides） were investigated using electrospray ionization mass spectrometry（ESI-MS） in the gas phase. The results of relative binding affinities in negative ion mode revealed that several factors impact on the duplexbinding properties of ginsenosides. Glycosylations of 20（S）-protopanaxadiol ginsenosides at the position C-20 and 20（S）-protopanaxatriol classification at the position C-6 enhanced the fraction of bound DNA sharply. A rhamnose moiety shows little lower binding intensities than glucose at the same position.Ginsenosides of 20（S）-protopanaxatriol result in subtle higher binding affinities toward the duplex DNA than 20（S）-protopanaxadiol family. However, glycosylation with two sugar moieties does not show a higher binding affinity than with only one moiety. The collision-induced dissociation experimental data demonstrate the gas-phase stability and fragmentation patterns of the ginsenoside/DNA complexes are related to the glycoside number. Positive ion ESI mass spectra of the complexes were also recorded. The result of ESI-MS suggests that hydrogen bonds are the dominate interaction between ginsenosides and DNA. Similar results were obtained in solution-phase by UV spectroscopy, which exhibit a hyperchromism and blue-shift effect when DNA solution was titrated by individual ginsenoside.
文摘基于国际生命条码联盟(CBOL,the Consortium for the Barcode of Life)提出的barcoding技术所确定的序列区域,针对猪的线粒体COⅠ基因序列设计特异性引物。为了避免食品中PCR抑制剂的影响,本实验设置GCG(胰岛素受体)基因125bp的纯化片段为内参照,控制假阴性结果的出现。通过优化PCR反应条件,猪和GCG基因的特异性产物在79.2℃和75℃有特异性熔解峰。设置牛、羊、鸡、兔、鼠和空白对照,均无扩增。测序结果表明,该猪特异性引物的PCR产物含有245bp的核苷酸序列与GenBank中相应序列吻合;该方法检出限为0.001%。
基金the National Natural Science Foundation of China,No.81672100 and No.81371894the Key Laboratory for Laboratory Medicine of Jiangsu Province of China,No.ZDXKB2016005Jiangsu Provincial Commission of Health and Family Planning,No.H201609
文摘BACKGROUND Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed. AIM To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method. METHODS A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay. RESULTS Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P<0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with markedsevere inflammation were significantly higher than those with mild-moderate inflammation (P<0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R^2=0.673, P<0.01) or plasma DNA (R^2=0.597, P<0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild–moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively. CONCLUSION The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.
基金the National Natural Science Foundation of China (No. 20075012) and the Outstanding Scholar Program of Nankai University.
文摘The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.
